Uptake and translocation of cations play essential roles in plant nutrition, signal transduction, growth, and development. Among them, potassium (K+) and sodium (Na+) have been the focus of numerous physiological studies because K+ is an essential macronutrient and the most abundant inorganic cation in plant cells, whereas Na+ toxicity is a principal component of the deleterious effects associated with salinity stress. Although the homeostasis of these two ions was long surmised to be fine tuned and under complex regulation, the myriad of candidate membrane transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless perplexing. Recent advances have shown that, in addition to their function in vacuolar accumulation of Na+, proteins of the NHX family are endosomal transporters that also play critical roles in K+ homeostasis, luminal pH control, and vesicle trafficking. The plasma membrane SOS1 protein from Arabidopsis thaliana, a highly specific Na+/H+ exchanger that catalyses Na+ efflux and that regulates its root/shoot distribution, has also revealed surprising interactions with K+ uptake mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely unknown but two CHX isoforms, AtCHX17 and AtCH23, have been shown to affect K+ homeostasis and the control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of cation exchangers are reviewed and discussed.

P1B-type ATPases transport a variety of metals (Cd2+, Zn2+, Pb2+, Co2+, Cu2+, Ag+, Cu+) across biomembranes. Characteristic sequences CP[C/H/S] in transmembrane fragment H6 were observed in the putative transporting metal site of the founding members of this subfamily (initially named CPx-ATPases). In spite of their importance for metal homeostasis and biotolerance, their mechanisms of ion selectivity are not understood. Studies of better-characterized P(II)-type ATPases (Ca-ATPase and Na,K-ATPase) have identified three transmembrane segments that participate in ion binding and transport. Testing the hypothesis that metal specificity is determined by conserved amino acids located in the equivalent transmembrane segments of P1B-type ATPases (H6, H7, and H8), 234 P1B-ATPase protein sequences were analyzed. This showed that although H6 contains characteristic CPX or XPC sequences, conserved amino acids in H7 and H8 provide signature sequences that predict the metal selectivity in each of five P1B-ATPase subgroups identified. These invariant amino acids contain diverse side chains (thiol, hydroxyl, carbonyl, amide, imidazolium) that can participate in transient metal coordination during transport and consequently determine the particular metal selectivity of each enzyme. Each subgroup shares additional structural characteristics such as the presence (or absence) of particular amino-terminal metal-binding domains and the number of putative transmembrane segments. These differences suggest unique functional characteristics for each subgroup in addition to their particular metal specificity.

Plants are sessile and therefore have developed mechanisms to adapt to their environment, including the soil mineral nutrient composition. Ionomics is a developing functional genomic strategy designed to rapidly identify the genes and gene networks involved in regulating how plants acquire and accumulate these mineral nutrients from the soil. Here, we report on the coupling of high-throughput elemental profiling of shoot tissue from various Arabidopsis accessions with DNA microarray-based bulk segregant analysis and reverse genetics, for the rapid identification of genes from wild populations of Arabidopsis that are involved in regulating how plants acquire and accumulate Na(+) from the soil. Elemental profiling of shoot tissue from 12 different Arabidopsis accessions revealed that two coastal populations of Arabidopsis collected from Tossa del Mar, Spain, and Tsu, Japan (Ts-1 and Tsu-1, respectively), accumulate higher shoot levels of Na(+) than do Col-0 and other accessions. We identify AtHKT1, known to encode a Na(+) transporter, as being the causal locus driving elevated shoot Na(+) in both Ts-1 and Tsu-1. Furthermore, we establish that a deletion in a tandem repeat sequence approximately 5 kb upstream of AtHKT1 is responsible for the reduced root expression of AtHKT1 observed in these accessions. Reciprocal grafting experiments establish that this loss of AtHKT1 expression in roots is responsible for elevated shoot Na(+). Interestingly, and in contrast to the hkt1-1 null mutant, under NaCl stress conditions, this novel AtHKT1 allele not only does not confer NaCl sensitivity but also cosegregates with elevated NaCl tolerance. We also present all our elemental profiling data in a new open access ionomics database, the Purdue Ionomics Information Management System (PiiMS; http://www.purdue.edu/dp/ionomics). Using DNA microarray-based genotyping has allowed us to rapidly identify AtHKT1 as the casual locus driving the natural variation in shoot Na(+) accumulation we observed in Ts-1 and Tsu-1. Such an approach overcomes the limitations imposed by a lack of established genetic markers in most Arabidopsis accessions and opens up a vast and tractable source of natural variation for the identification of gene function not only in ionomics but also in many other biological processes.

Fluorescence imaging is perhaps the most powerful technique currently available for continuously observing the dynamic intracellular biochemistry of single living cells. However, fluorescent indicator dyes have been available only for simple inorganic ions such as Ca2+, H+, Na+, K+, Mg2+ and Cl-. We now report a fluorescent indicator for the adenosine 3',5'-cyclic monophosphate (cAMP) signalling pathway. The sensor consists of cAMP-dependent protein kinase in which the catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye such as fluorescein or rhodamine capable of fluorescence resonance energy transfer in the holoenzyme complex R2C2. When cAMP molecules bind to the R subunits, the C subunits dissociate, thereby eliminating energy transfer. The change in shape of the fluorescence emission spectrum allows cAMP concentrations and the activation of the kinase to be nondestructively visualized in single living cells microinjected with the labelled holoenzyme.

The recent determination of the crystal structure of the leucine transporter from Aquifex aeolicus (aaLeuT) has provided significant insights into the function of neurotransmitter:sodium symporters. Transport by aaLeuT is Cl(-) independent, whereas many neurotransmitter:sodium symporters from higher organisms depend on Cl(-) ions. However, the only Cl(-) ion identified in the aaLeuT structure interacts with nonconserved residues in extracellular loops, and thus the relevance of this binding site is unclear. Here, we use calculations of pK(A)s and homology modeling to predict the location of a functionally important Cl(-) binding site in serotonin transporter and other Cl(-)-dependent transporters. We validate our model through the site-directed mutagenesis of residues predicted to coordinate the Cl(-) ion and through the observation of sequence conservation patterns in other Cl(-)-dependent transporters. The proposed site is located midway across the membrane and is formed by residues from transmembrane helices 2, 6, and 7. It is close to the NaNa(+) ions during transport. Other implications of the model are also discussed.

Calmodulin kinase (CaMK) II is linked to arrhythmia mechanisms in cellular models where repolarization is prolonged. CaMKII upregulation and prolonged repolarization are general features of cardiomyopathy, but the role of CaMKII in arrhythmias in cardiomyopathy is unknown.

We studied a mouse model of cardiac hypertrophy attributable to transgenic (TG) overexpression of a constitutively active form of CaMKIV that also has increased endogenous CaMKII activity. ECG-telemetered TG mice had significantly more arrhythmias than wild-type (WT) littermate controls at baseline, and arrhythmias were additionally increased by isoproterenol. Arrhythmias were significantly suppressed by an inhibitory agent targeting endogenous CaMKII. TG mice had longer QT intervals and action potential durations than WT mice, and TG cardiomyocytes had frequent early afterdepolarizations (EADs), a hypothesized mechanism for triggering arrhythmias. EADs were absent in WT cells before and after isoproterenol, whereas EAD frequency was unaffected by isoproterenol in TG mice. L-type Ca2+ channels (LTTCs) can activate EADs, and LTCC opening probability (Po) was significantly higher in TG than WT cardiomyocytes before and after isoproterenol. A CaMKII inhibitory peptide equalized TG and WT LTCC Po and eliminated EADs, whereas a peptide antagonist of the Na+/Ca2+ exchanger current, also hypothesized to support EADs, was ineffective.

These findings support the hypothesis that CaMKII is a proarrhythmic signaling molecule in cardiac hypertrophy in vivo. Cellular studies point to EADs as a triggering mechanism for arrhythmias but suggest that the increase in arrhythmias after beta-adrenergic stimulation is independent of enhanced EAD frequency.

After chronic spinal cord injury motoneurons exhibit large plateau potentials (sustained depolarizations triggered by brief inputs) that play a primary role in the development of muscle spasms and spasticity (Bennett et al. 2001a,b). The present study examined the voltage-gated persistent inward currents (PICs) underlying these plateaus. Adult rats were spinalized at the S2 sacral spinal level and after 2 mo, when spasticity developed, intracellular recordings were made from motoneurons below the injury. For recording, the whole sacrocaudal spinal cord was removed and maintained in vitro in normal artificial cerebral spinal fluid (nACSF), without application of neuromodulators. During a slow triangular voltage-clamp command (ramp) a PIC was activated with a threshold of -54.2 +/- 4.8 mV (similar to plateau threshold), with a peak current of 2.88 +/- 0.95 nA and produced a pronounced negative-slope region in the V-I relation. This PIC was in part mediated by Cav1.3 L-type calcium channels because it was low threshold and significantly reduced by 10 to 20 microM nimodipine or 400 microM Cd2+. The PIC that remained during a calcium channel blockade (in Cd2+) was completely and rapidly blocked by tetrodotoxin (TTX; 0.5 to 2 microM), and thus was a TTX-sensitive persistent sodium current. This persistent sodium current was activated rapidly about 7 mV below the spike threshold (spike threshold -46.1 +/- 4.5 mV), contributed approximately 1/2 of the initial peak of the total PIC, inactivated partly to contribute only approximately 1/3 of the sustained PIC (at 5 to 10 s), and deactivated rapidly with hyperpolarization (<50 ms). When TTX was added to the bath first, the nimodipine-sensitive persistent calcium current (L-type) was seen in isolation; it was slowly activated (>250 ms), had a low but variable threshold (either slightly above or below the spike threshold), contributed the other approximately 1/2 of the initial peak of the total PIC (before TTX), did not usually inactivate with time (contributed approximately two-thirds of the sustained PIC), and deactivated slowly with hyperpolarization to rest (in >300 ms). In summary, low-threshold persistent calcium (Cav1.3) and sodium currents spontaneously develop in motoneurons of chronic spinal rats and these enable large, rapidly activated plateaus that ultimately lead to spasticity.

Arterial pulse wave velocity, an established index of arterial distensibility, was measured together with arterial pressure in a group of 524 normal subjects of both sexes 2 months to 94 years old (mean age 45.6 +/- 15.3 years [SD]) in rural Guangzhou, China, an area with known low prevalence of hypertension. Fasting serum lipid levels and overnight Na+ and K+ urinary excretion levels were determined in a subgroup of 104 subjects (ages 8 to 88 years). Comparisons were made with data obtained similarly from normal subjects in urban Beijing, an area with known high prevalence of hypertension. Serum cholesterol levels were similar and low in each group (Guangzhou, 4.34 +/- 0.12 mmol/liter [SE]; BEijing, 4.49 +/- 0.11 mmol/liter). Prevalence of hypertension (WHO criteria) was 4.9% (Guangzhou) and 15.6% (Beijing). In Guangzhou subjects pulse wave velocity was consistently lower in the aorta, arm, and leg, and increased to a lesser degree with age compared with Beijing subjects. Regression equations (x = pulse wave velocity [cm/sec], y = age [years]) were as follows: (1) aorta, Guangzhou: y = 5.1x + 533, r = .552, p less than .05; Beijing: y = 9.2x + 615, r = .673, p less than .001; (2) arm, Guangzhou: y = 0.61x + 817, r = .121, p less than .05; Beijing: y = 4.8x + 998, r = .453, p less than .001; (3) leg, Guangzhou: y = 4.43x + 718, r = .512, p less than .05; Beijing: y = 5.6x + 791, r = .630, p less than .001.(ABSTRACT TRUNCATED AT 250 WORDS)

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159-212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487-517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816-824). When activated, they produce depolarization through the influx of Na+, but their high Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience. 55:1015-1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525-3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of approximately 30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin.

Previous work has suggested that a Na-Ca exchanger may have a key role in visual transduction in retinal rods. This exchanger is thought to maintain a low internal free Ca2+ concentration in darkness and to contribute to the rod's recovery after light by removing any internally released Ca2+. Little else is known about this transport mechanism in rods. We describe here an inward membrane current recorded from single isolated rods which appears to be associated with such external Na+-dependent Ca2+ efflux activity. External Na+, but not Li+, could generate this current; high external K+ inhibited it while small amounts of La3+ (10 microM) completely abolished it. The exchanger can also transport Sr2+, but not Ba2+ or other divalent cations. The exchange ratio was estimated to be 3Na+:1Ca2+. As well as demonstrating clearly the Na-Ca exchanger in the rod outer segment, our experiments also cast serious doubt on the commonly held view that light simply releases internal Ca2+ to bind to and block the light-sensitive conductance.

OBJECTIVE

The hypothesis that an increased or prolonged Ca2+ transient during an abbreviated action potential can give rise to early afterdepolarizations (EADs) and triggered arrhythmia by enhanced forward sodium-calcium (Na-Ca) exchange was examined.

BACKGROUND

Because pulmonary veins have the shortest action potential of any cardiac tissue, we examined this hypothesis in canine pulmonary vein sleeves during interventions further shortening the action potential and increasing the calcium transient.

METHODS

Extracellular bipolar electrode, intracellular microelectrode, and isometric force (a surrogate marker for the Ca2+ transient) recordings were obtained from superfused canine pulmonary veins.

RESULTS

An elevation and prolongation of the terminal phase of repolarization (EADs) were observed during interventions increasing contractile force; isoproterenol or norepinephrine (3.2 x 10(-11) to 3.2 x 10(-7)M), hypothermia, and pacing (post-extrasystolic potentiation, post-pacing pause). The EAD formation was prevented by ryanodine (10 microM) or reversed by transiently increasing [Ca2+](o) from 1.35 to 5 mM (inhibition of forward Na-Ca exchange). Pacing-induced EADs were enhanced by re-introduction of normal Tyrode solution (Na+ = 130 mM) after substitution of 30 mM NaCl with 30 mM LiCl (stimulation of forward Na-Ca exchange). With norepinephrine or isoproterenol (3.2 x 10(-8)M) + acetylcholine (10(-7)M) (to enhance the Ca2+ transient and further shorten the abbreviated action potential, respectively), tachycardia-pause initiated arrhythmia (1,132 +/- 153 beats/min) lasting >1 s was observed. Rapid firing was prevented by either suppression of the Ca2+ transient (ryanodine) or transiently increasing [Ca2+](o).

CONCLUSIONS

The data show EAD formation in superfused canine pulmonary veins, enhanced by an increased Ca2+ transient and increased Na-Ca exchange current. With subsequent shortening of the action potential with acetylcholine, tachycardia-pause triggers rapid firing within the PV sleeve.

The Arabidopsis thaliana vacuolar Na+/H+ antiporter AtNHX1 is a salt tolerance determinant. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in AtNHX1 and prototypical mammalian NHE Na+/H+ exchangers led to the identification of five additional AtNHX genes (AtNHX2-6). The AtNHX1 and AtNHX2 mRNAs are the most prevalent transcripts among this family of genes in seedling shoots and roots. A lower-abundance AtNHX5 mRNA is present in both shoots and roots, whereas AtNHX3 transcript is expressed predominantly in roots. AtNHX4 and AtNHX6 mRNAs were detected only by RT-PCR. AtNHX1, 2 or 5 suppress, with differential efficacy, the Na+/Li+-sensitive phenotype of a yeast mutant that is deficient in the endosomal/vacuolar Na+/H+ antiporter ScNHX1. Ion accumulation data indicate that these AtNHX proteins function to facilitate Na+ ion compartmentalization and maintain intracellular K+ status. Seedling steady-state mRNA levels of AtNHX1 and AtNHX2 increase similarly after treatment with NaCl, an equi-osmolar concentration of sorbitol, or ABA, whereas AtNHX5 transcript abundance increases only in response to salt treatment. Hyper-osmotic up-regulation of AtNHX1, 2 or 5 expression is not dependent on the SOS pathway that controls ion homeostasis. However, steady-state AtNHX1, 2 and 5 transcript abundance is greater in sos1, sos2 and sos3 plants growing in medium that is not supplemented with sorbitol or NaCl, providing evidence that transcription of these genes is negatively affected by the SOS pathway in the absence of stress. AtNHX1 and AtNHX2 transcripts accumulate in response to ABA but not to NaCl in the aba2-1, mutant indicating that the osmotic responsiveness of these genes is ABA-dependent. An as yet undefined stress signal pathway that is ABA- and SOS-independent apparently controls transcriptional up-regulation of AtNHX5 expression by hyper-saline shock. Similar to AtNHX1, AtNHX2 is localized to the tonoplast of plant cells. Together, these results implicate AtNHX2 and 5, together with AtNHX1, as salt tolerance determinants, and indicate that AtNHX2 has a major function in vacuolar compartmentalization of Na+.

Pandemic influenza requires interspecies transmission of an influenza virus with a novel hemagglutinin (HA) subtytpe that can adapt to its new host through either reassortment or point mutations and transmit by aerosolized respiratory droplets. Two previous pandemics of 1957 and 1968 resulted from the reassortment of low pathogenic avian viruses and human subtypes of that period; however, conditions leading to a pandemic virus are still poorly understood. Given the endemic situation of avian H9N2 influenza with human-like receptor specificity in Eurasia and its occasional transmission to humans and pigs, we wanted to determine whether an avian-human H9N2 reassortant could gain respiratory transmission in a mammalian animal model, the ferret. Here we show that following adaptation in the ferret, a reassortant virus carrying the surface proteins of an avian H9N2 in a human H3N2 backbone can transmit efficiently via respiratory droplets, creating a clinical infection similar to human influenza infections. Minimal changes at the protein level were found in this virus capable of respiratory droplet transmission. A reassortant virus expressing only the HA and neuraminidase (NA) of the ferret-adapted virus was able to account for the transmissibility, suggesting that currently circulating avian H9N2 viruses require little adaptation in mammals following acquisition of all human virus internal genes through reassortment. Hemagglutinin inhibition (HI) analysis showed changes in the antigenic profile of the virus, which carries profound implications for vaccine seed stock preparation against avian H9N2 influenza. This report illustrates that aerosolized respiratory transmission is not exclusive to current human H1, H2, and H3 influenza subtypes.

BACKGROUND

In the failing human heart, sarcoplasmic reticulum (SR) calcium handling is impaired, and therefore, calcium elimination and diastolic function may depend on the expression of sarcolemmal Na+-Ca2+ exchanger.

RESULTS

Force-frequency relations were studied in ventricular muscle strip preparations from failing human hearts (n=29). Protein levels of Na+-Ca2+ exchanger and SR Ca2+-ATPase were measured in the same hearts. Hearts were divided into 3 groups by discriminant analysis according to the behavior of diastolic function when stimulation rate of muscle strips was increased from 30 to 180 min-1. At 180 compared with 30 min-1, diastolic force was increased by 160%, maximum rate of force decline was decreased by 46%, and relaxation time was unchanged in group III. In contrast, in group I, diastolic force and maximum rate of force decline did not change, and relaxation time decreased by 20%. Na+-Ca2+ exchanger was 66% higher in group I than in group III. Na+-Ca2+ exchanger was inversely correlated with the frequency-dependent rise of diastolic force when stimulation rate was increased (r=-0.74; P<0.001). Compared with nonfailing human hearts (n=6), SR Ca2+-ATPase was decreased and Na+-Ca2+ exchanger unchanged in group III, whereas Na+-Ca2+ exchanger was increased and SR Ca2+-ATPase unchanged in group I. Results with group II hearts were between those of group I and group III hearts.

CONCLUSIONS

By discriminating failing human hearts according to their diastolic function, we identified different phenotypes. Disturbed diastolic function occurs in hearts with decreased SR Ca2+-ATPase and unchanged Na+-Ca2+ exchanger, whereas increased expression of the Na+-Ca2+ exchanger is associated with preserved diastolic function.

Most organs consist of networks of interconnected tubes that serve as conduits to transport fluid and cells and act as physiological barriers between compartments. Biological tubes are assembled through very diverse developmental processes that generate structures of different shapes and sizes. Nevertheless, all biological tubes invariably possess one single lumen. The mechanisms responsible for single lumen specification are not known. Here we show that zebrafish mutants for the MODY5 and familial GCKD gene tcf2 (also known as vhnf1) fail to specify a single lumen in their gut tube and instead develop multiple lumens. We show that Tcf2 controls single lumen formation by regulating claudin15 and Na+/K+-ATPase expression. Our in vivo and in vitro results indicate that Claudin15 functions in paracellular ion transport to specify single lumen formation. This work shows that single lumen formation is genetically controlled and appears to be driven by the accumulation of fluid.

The skin interstitium sequesters excess Na+ and Cl- in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl- accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na+, Cl-, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure-regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3-mediated modification of cutaneous lymphatic capillary function.

The search for natriuretic hormones or factors by studies of negative pressure breathing, atrial distension experiments, head-out water immersion, expansion of blood volume, Na+/K+-ATPase inhibitors and parabiosis experiments in Dahl rats has led to the finding that the atria are a peptide-secreting endocrine gland. This new natriuretic hormone has now been purified, sequenced and synthetized, and its cDNA and gene have been cloned. The native and synthetic hormones exert identical wide ranging effects (possibly through particulate guanylate cyclase stimulation and adenylate cyclase inhibition) on the kidney, blood vessels, adrenal cortex, and pituitary. Physiopathologic implications of the hormone in experimental hypertension, congestive heart failure, and expansion of blood volume are beginning to emerge.

1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole-cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti-influenza drug rimantadine. 2. The ion selectivity of the rimantadine-sensitive current through M2 was determined. Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl- concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10(-7). 3. The M2 conductance increased as external pH decreased below 8.5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 microM and 1 microM, respectively, under physiological conditions. 4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans-Golgi pH during virus infection.

Studies have been carried out on the movement of salt and water across the small intestine of the rat. Segments of the ileum of anesthetized rats have been perfused in vivo with unbuffered NaCl solutions or isotonic solutions of NaCl and mannitol. Kinetic analysis of movements of Na(24) and Cl(36) has permitted determination of the efflux and influx of Na and Cl. Net water absorption has been measured using hemoglobin as a reference substance. Water was found to move freely in response to gradients of osmotic pressure. Net water flux from isotonic solutions with varying NaCl concentration was directly dependent on net solute flux. The amount of water absorbed was equivalent to the amount required to maintain the absorbed solute at isotonic concentration. These results have been interpreted as indicating that water movement is a passive process depending on gradients of water activity and on the rate of absorption of solute. The effluxes of Na and Cl are linear functions of concentration in the lumen, but both ions are actively transported by the ileum according to the criterion of Ussing (Acta Physiol. Scand., 1949, 19, 43). The electrical potential difference between the lumen and plasma has been interpreted as a diffusion potential slightly modified by the excess of active Cl flux over active Na flux. The physical properties of the epithelial membrane indicate that it is equivalent to a membrane having negatively charged uniform right circular pores of 36 A radius occupying 0.001 per cent of the surface area.

1. Sodium was injected into an identified snail neurone by passing current between two intracellular micro-electrodes, the membrane potential being recorded with a third micro-electrode.2. The injection of about 25 p-equiv Na, but not the injection of similar quantities of K or Li, caused a hyperpolarization of up to 20 mV. This response to Na injection was blocked by application of ouabain or removal of external K, indicating that it was due to the stimulation of an electrogenic pump.3. To measure the current produced by the sodium pump the output of a feed-back amplifier was fed into the cell via a fourth intracellular micro-electrode so as to keep the average membrane potential constant. The pump current, measured in this way, rose at a constant rate during, and declined exponentially after, an injection of Na, the decline having an average time constant of 4.4 min. The total charge transferred by the pump was between a third and a quarter of the charge passed to inject sodium.4. An intracellular Na-sensitive glass micro-electrode was used to follow changes in the concentration of intracellular Na ions. The results showed that both the pump current and the rate of Na extrusion were proportional to the concentration of intracellular Na ions above the normal level.5. It was concluded that about two thirds of the Na extruded was coupled to the active transport of other ions, probably to the uptake of K, the uncoupled third producing the electrogenic effect.

The drive for food is one of the most powerful of human and animal behaviors. Dopamine, a neurotransmitter involved with motivation and reward, its believed to regulate food intake in laboratory animals by modulating its rewarding effects through the nucleus accumbens (NA). Here we assess the involvement of dopamine in "nonhedonic" food motivation in humans. Changes in extracellular dopamine in striatum in response to nonhedonic food stimulation (display of food without consumption) were evaluated in 10 food-deprived subjects (16-20 h) using positron emission tomography (PET) and [11C]raclopride (a D2 receptor radioligand that competes with endogenous dopamine for binding to the receptor). To amplify the dopamine changes we pretreated subjects with methylphenidate (20 mg p.o.), a drug that blocks dopamine transporters (mechanism for removal of extracellular dopamine). Although the food stimulation when preceded by placebo did not increase dopamine or the desire for food, the food stimulation when preceded by methylphenidate (20 mg p.o.) did. The increases in extracellular dopamine were significant in dorsal (P < 0.005) but not in ventral striatum (area that included NA) and were significantly correlated with the increases in self-reports of hunger and desire for food (P < 0.01). These results provide the first evidence that dopamine in the dorsal striatum is involved in food motivation in humans that is distinct from its role in regulating reward through the NA. In addition it demonstrates the ability of methylphenidate to amplify weak dopamine signals.

The rat thyroid Na+/I- symporter (NIS) was expressed in Xenopus laevis oocytes and characterized using electrophysiological, tracer uptake, and electron microscopic methods. NIS activity was found to be electrogenic and Na+-dependent (Na+>>) Li+>>) H+). The apparent affinity constants for Na+ and I- were 28 +/- 3 mM and 33 +/- 9 microM, respectively. Stoichiometry of Na+/anion cotransport was 2:1. NIS was capable of transporting a wide variety of anions (I-, ClO3-, SCN-, SeCN-, NO3-, Br-, BF4-, IO4-, BrO3-, but perchlorate (ClO4-) was not transported. In the absence of anion substrate, NIS exhibited a Na+-dependent leak current (approximately 35% of maximum substrate-induced current) with an apparent Na+ affinity of 74 +/- 14 mM and a Hill coefficient (n) of 1. In response to step voltage changes, NIS exhibited current transients that relaxed with a time constant of 8-14 ms. Presteady-state charge movements (integral of the current transients) versus voltage relations obey a Boltzmann relation. The voltage for half-maximal charge translocation (V0.5) was -15 +/- 3 mV, and the apparent valence of the movable charge was 1. Total charge was insensitive to [Na+]o, but V0.5 shifted to more negative potentials as [Na+]o was reduced. NIS charge movements are attributed to the conformational changes of the empty transporter within the membrane electric field. The turnover rate of NIS was>>/=22 s-1 in the Na+ uniport mode and>>/=36 s-1 in the Na+/I- cotransport mode. Transporter density in the plasma membrane was determined using freeze-fracture electron microscopy. Expression of NIS in oocytes led to a approximately 2. 5-fold increase in the density of plasma membrane protoplasmic face intramembrane particles. On the basis of the kinetic results, we propose an ordered simultaneous transport mechanism in which the binding of Na+ to NIS occurs first.

Catecholamine secretory vesicle core proteins (chromogranins) contain an activity that inhibits catecholamine release, but the identity of the responsible peptide has been elusive. Size-fractionated chromogranins antagonized nicotinic cholinergic-stimulated catecholamine secretion; the inhibitor was enriched in processed chromogranin fragments, and was liberated from purified chromogranin A. Of 15 synthetic peptides spanning approximately 80% of chromogranin A, one (bovine chromogranin A344-364 [RSMRLSFRARGYGFRGPGLQL], or catestatin) was a potent, dose-dependent (IC50 approximately 200 nM), reversible secretory inhibitor on pheochromocytoma and adrenal chromaffin cells, as well as noradrenergic neurites. An antibody directed against this peptide blocked the inhibitory effect of chromogranin A proteolytic fragments on nicotinic-stimulated catecholamine secretion. This region of chromogranin A is extensively processed within chromaffin vesicles in vivo. The inhibitory effect was specific for nicotinic cholinergic stimulation of catecholamine release, and was shared by this chromogranin A region from several species. Nicotinic cationic (Na+, Ca2+) signal transduction was specifically disrupted by catestatin. Even high-dose nicotine failed to overcome the inhibition, suggesting noncompetitive nicotinic antagonism. This small domain within chromogranin A may contribute to a novel, autocrine, homeostatic (negative-feedback) mechanism controlling catecholamine release from chromaffin cells and neurons.

Following the experimental findings of Atwater et al. (In Biochemistry Biophysics of the Pancreatic-beta-Cell, George Thieme Verlag, New York, 100-107), we have formulated a mathematical model for ionic and electrical events that take place in pancreatic-beta-cells. Our formulation incorporates a Hodgkin-Huxley type gating mechanism for Ca2+ and K+ channels, in addition to Ca2+ gated K+-channels. Consistent with the experimental observations, our model generates spikes and bursts in beta-cell membrane potentials and gives the correct responses to additions of glucose, quinine, and tetraethylammonium ions. The response of the oscillations to ouabain and changing concentrations of external K+ can be incorporated into the present model, although a more complete treatment would require inclusion of the Na+/K+ pump.